Two-dimensional slab gel electrophoresis is still the most used approach to proteomics and it might be still for several years, if other limitations still present are addressed. Indeed, this remains a time-consuming and laborious procedure, requiring trained personnel, on the hands of whom the quality of results is mainly depending. Although the post-electrophoretic steps are highly robotized, the separation step is far from it, so that problems with accuracy and consistency can arise from variations in the numerous parameters to keep under control. Some of these are for example, sample loading and rehydration, in terms of sample amount, losses, and homogeneity of the strip, strip handling with risk of damaging and contamination, imprecise and slow coupling of the strip to the gel, gel casting and polymerization, in terms of homogeneity, casting and reaction speed, especially for gradients, air sensitivity, time for completion until run is started, risk to trap bubbles causing consequently also field discontinuities, increase in temperature during the run, pH and viscosity changes, and loss of buffer capacity. Lack of acceptable reproducibility, meaning that no two gel images are directly superimposable, remains therefore a major problem if considered that gels are mostly made to be compared, e.g., to detect and quantify differences in protein expression between experimental pairs of complex protein samples, i.e., each sample having more than 10 individual protein components. In practical terms, this translates in the need to run more gels to build reference maps for each condition and reach a certain degree of certainty, which in turn means even more manual work.
A technique, apparently overcoming this problem, was introduced in 1997, namely fluorescent 2-D differential gel electrophoresis (DIGE) by Unlu et al. (Unlu M., Morgan M. E., Minden J. S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis, 18:2071-2077). DIGE is based on the use of two mass- and charge-matched N-hydroxy succinimidyl ester derivatives of the fluorescent cyanin dyes Cy3 and Cy5, possessing distinct excitation and emission spectra, to differentially label lysine residues of two protein samples, which are then mixed and run on the same gel. Thus, matching is automatic and straightforward and in principle only a single gel could be sufficient. However, for a proper statistical evaluation, at least three to five gels are required as well. To make things even more complicated than before is the fact that very stringent labeling conditions should be followed. It is indeed well known that pre-labeling can generate a large number of positional isomers as well as partially reacted species yielding very heterogeneous results. Labeling must be therefore minimal, trying to achieve possibly the addition to a single lysine residue on the entire protein molecule. In addition, the over-reacted species might precipitate as a result of an acquired increased hydrophobicity, but the biggest issue is the fact that one cannot simply run a DIGE gel and cut out the spots of the differentially expressed proteins for subsequent mass spectroscopy (MS) analysis. Indeed, there is no way to predict to which lysine, thus to which peptide of the digested spot, the covalent fluorescent label will be attached, so that peptide identification might be problematic. Moreover, after the gel has been removed from the fluorescence scanner, the spots will no longer be visible, so that a protein dye or other visual staining technique should be thus used anyway for the post-electrophoretic visualization. Finally, perhaps the biggest limitation is represented by the very high cost of the equipment, software and reagents.
Automation associated with better reproducibility are the main strengths of the instrumental chromatographic approach, as no further manual intervention is required after the sample has been loaded. Nevertheless, this is true only when using the same column and running the same method sample after sample in a sequential order. Columns of the same size packed with the same material might give indeed different elution times, as column packing is per se not perfectly reproducible. New materials such as, e.g., monoliths, bring with them new advantages but columns are still made one by one, meaning that, in analogy to gels, no two chromatograms run in parallel are superimposable. Besides this, limitations due to cost and complexity of instrumentation make this approach after all not faster and not really more convenient, despite other inherent advantages like on-line detection and the possibility of direct coupling to MS. Gels, on the other hand, can be easily run in parallel, can offer under optimal conditions superior resolution, and can be directly compared by imaging. The potential is therefore still very big if integration and automation, thus higher reproducibility and throughput, are achieved for gels too, allowing to run more and comparable gels in less time with less work and reduced costs.